Method and apparatus for quantitating enzyme activity

ABSTRACT

Method and apparatus for quantitating enzyme activity in blood and other fluids, using a spot test technique. The test utilizes enzyme standards freeze-dried in absorbent discs held under a membrane filter. Other discs above the filter contain substrate, cofactors, and indicators. The spot test plates are entirely self-contained, requiring neither instrumentation nor measurement of fluid volumes, and the test can be carried out under any ambient conditions. The test is particularly applicable to emergency screening requirements, such as may occur in hospital receiving rooms, doctors&#39;&#39; offices, ambulances, or whereever rapid diagnosis of myocardial infraction may be required.

United States Patent Moyer et al.

[15] 3,663,374 [4 1 May 16, 1972 METHOD AND APPARATUS FOR QUANTITATIN GENZYME ACTIVITY [72] inventors: Rudolph H. Moyer, West Covina; Donald J.Sibbett, Cucamonga, both of Calif. [73] Assignee: Geomet, Incorporated,Rockville, Md. [22] Filed: Aug. 14, 1970 [21 Appl. No.: 63,842

[52] US. Cl. ..195/103.5 R, 195/99 [51} lnt.Cl. ..G0ln 31/14 [58] FieldofSearch ..l95/103.5, 99; 23/253 TP, 230 B,

I S I, 1 References Cited UNITED STATES PATENTS 3,526,480 9/1970 Findlet al. ..23/253 TP X 3,367,841 2/1968 Buissiere et a1. ..195/103.5 R

3,261,668 7/1966 Natelson ..23/253 TP Primary Examiner-A. Louis MonacellAssistant Examiner-Max D. Hensley Att0rney-David H. Semmes [5 7]ABSTRACT Method and apparatus for quantitating enzyme activity in bloodand other fluids, using a spot test technique. The test utilizes enzymestandards freeze-dried in absorbent discs held under a membrane filter.Other discs above the filter contain substrate, cofactors, andindicators. The spot test plates are entirely self-contained, requiringneither instrumentation nor measurement of fluid volumes, and the testcan be carried out under any ambient conditions. The test isparticularly applicable to emergency screening requirements. such as mayoccur in hospital receiving rooms, doctors offices, ambulances. orwhereever rapid diagnosis of myocardial infraction may be required.

10 Claims, 1 Drawing Figure Patented May 16, 1972 3,663,374

INVENTORS RUDOLPH H. MOYER DONALD J. SIBBETT ATTORNEY BACKGROUND OF THEINVENTION The use of blood enzymes for diagnosis of various pathologicalconditions is such standard clinical practice that test kits andautomated procedures are available for assay of most enzymes ofinterest. Myocardial infarction is one of the conditions eitherdiagnosed or verified through measurement of the level of activity ofseveral enzymes which escape into the bloodstream through damage to themyocardium.

Activity measurements of a combination of five enzymes are commonlyemployed for such diagnoses, and elevated levels of any of the followingin blood serum may be indicative of myocardial infarction:

Creatine phosphokinase (CPK) Lactate dehydrogenase (LDH)a-hydroxybutyrate dehydrogenase (HBD) Glutamic-oxalacetic transaminase(GOT) Glutamic-pyruvic transaminase (GPT) The activity of these enzymesin serum is usually determined by a procedure in which a product of thereaction is linked to the oxidation or reduction of nicotinamide-adeninedinucleotide, a process which can be followed spectrophotometrically.Commercially available reagent kits for assay of most of these enzymesrequire only the addition of a small quantity of blood serum and briefpreincubation prior to spectrophotometric determination of reactionrate. While the kits are convenient for use in a clinical laboratory,they require temperature control, the use of serum or plasma rather thanwhole blood, and the availability of a spectrophotometer. Because ofthese requirements, the usual tests are not well-suited for use inemergency situations, where the necessary instrumentation and trainedpersonnel may not be readily available.

SUMMARY OF THE INVENTION The method and apparatus utilize a simple spottest procedure in which the rate of color development on referencespots, containing pre-standardized increments of enzyme, is compareddirectly with that on a test spot where the enzyme extracted from bloodreacts. Direct comparison of the blood with reference spots eliminatesthe need for both temperature control and timing of reaction rates. Thetest measures enzyme activity of whole blood, and it therefore requiresneither instrumentation or operator skill. It can, therefore, beemployed for rapid screening in emergency situations where clinical testequipment and trained personnel may not be available.

An illustrative embodiment of the apparatus and a working embodiment ofa practical method will be explained with reference to the accompanyingdrawing in which the single figure is:

An expanded view of a test screening plate and components utilizedtherewith.

In one form of the screening test a plate format is used, as shown inthe drawing. This test measures the activity of creatine phosphokinase(CPK), a preferred indicator enzyme for early diagnosis of myocardialinfarction. The test is based on a reaction sequence which linksadenosine triphosphate (ATP), a product of the CPK reaction to reductionof nitro blue tetrazoleum for visualization of the extent of reaction ina given time interval. All reagents, along with the CPK standards, arefreeze-dried in discs of absorbent material which are held between twomatching polystyrene slides or plates as will be described in detailhereinafter with reference to the drawings.

The reagent system is similar to that routinely employed for CPK assayand is coupled to reduction of nitro blue tetrazoleum through theelectron carrier, N-methylphenazonium methosulfate. Concentrations ofreagents are adjusted to provide optimum reaction conditions when thefreeze-dried components are reconstituted by wetting the discs. Thefollowing reagents and concentrations, described in appropriateliterature, Nielsen, L. and Ludvigsen, B., J. Lab & Clin. Med., 62, -168(1963), and Rosalki, S.B., Nature, 207, 414 (1965), were selected asbeing appropriate.

Concentration (mg/ml.)

o s w-as O O O O O O .1 lU .0 lU

As required These reagents, except cystein hydrochloride, are dissolvedin glycyl-glycine buffer, adjusted to pH 6.8 and appropriatelydistributed on discs incorporated in the test apparatus structures foreffecting the testing.

An enzyme screening plate for practicing the invention is shown inexpanded or exploded view in the FIGURE and consists of upper slide 10and lower slide 12 of appropriate material such as the usual laboratorypolystyrene. Each of the slides has a plurality of openings or holes 14therethrough, in the disclosed structure being five in number forpurposes hereinafter to be described. Glass fiber discs 16 and 18 areadapted for sandwiching placement over the openings and held thereon bymeans of a retaining ring 20 of suitable material and in an appropriatemanner. The relative sizes of the glass fiber discs 16, 18 and retainingring 20, with respect to the hole 14 are indicated by appropriatelyplaced dashed lines at 22. Intermediate the slides 10 and 12, andaccordingly beneath the opening 14 in upper slide 10 and above hold 14in the lower slide 12, adapted for sandwiched positionment, are amembrane filter 24 and a cellulose acetate disc 26, the size thereofrelative to the holes 14 being indicated by broken lines at 28. Therelative sizes permit positionment and retaining of these variousaforesaid discs in conjunction with the retaining ring 20.

The distribution of the reagents referred to above on the various discsis as follows:

16 Upper glass fiber cysteine hydrochloride 18 Lower glass fibercomposite of all except PMS and enzymes 24 Membrane filter PMS 26Cellulose acetate CPK, Hex, G-6-P deH This separation of mutuallyincompatible reagents provides maximum storage stability and testreliability.

The glass fiber discs, besides serving as a reagent reservoir, removewhite blood cells to prevent clogging of the membrane filter whichremoves red cells and other particles. Thus, a drop of blood placed onthe upper glass fiber disc 16, takes up cysteine hydrochloride, reagentsfrom the lower disc 18, and PMS from the membrane filter 24, deliveringboth filtered plasma and reagents to the enzymes on the celluloseacetate 26 where the reaction takes place.

In practice, the reagents are applied as indicated above andfreeze-dried. The two slides are then sealed together, packaged, andstored. In the preferred configuration, five sets of discs are used toprovide five test spots. Spots A, B, and C contain CPK standardsrepresenting normal, elevated, and high enzyme levels, respectively.Spot D is a blank and contains all reagents except creatine phosphate.Spot E is for assay of CPK and contains reagents for the completereaction sequence. Obviously any desired arrangement or selection ofspots can be used.

In use, the slide is positioned with the retaining ring and glass fiberdiscs uppermost. A drop of water is applied to spots A, B, and C, a dropof whole blood to each of D and E. Since fluid uptake is governed by theabsorbency of the discs, the volume applied to each spot is of noimportance. The slide is then inverted and the development of blue colorobserved. After a few minutes, the intensity of color in spot E iscompared with the standards as indicated at spots A, B, and C for adirect reading of CPK activity.

Slides prepared as described above, and employed to discriminate betweennormal and elevated blood CPK give results which indicate that in use:

1 Operator skill is not a necessity.

2. Whole blood can be used directly.

3. The test clearly discriminates between normal and elevated CPKlevels.

4. Auxiliary instrumentation is not required.

5. Measurement of fluid volumes is unnecessary.

6. lntemal standards eliminate the need for both temperature control andtiming of the reaction.

Accordingly, the spot test in accordance with the invention is a usefultool for screening patients for myocardial infarction in emergencysituations.

This system may also be utilized for screening the blood for elevatedlevels of other enzymes which are released into the blood duringmyocardial infarction: lactate dehydrogenase, ahydroxybutyratedehydrogenase, glutamic-oxalacetic transaminase, and glutamic-pyruvictransaminase. Appropriate modifications of the reagent system containedin the plate assembly is required in these cases.

Manifestly minor changes in the structure and test procedure can beeffected without departing from the spirit and scope of the invention asdefined in and limited solely by the appended claims.

We claim:

1. In a system for quantitating enzyme activity in whole blood:

A. means defining a plurality of separate restricted test zone areas ina rigid array;

B. said areas including in a rigidly confined-stacked array a pluralityof superposed test reagent impregnated members in a rigidly confinedcolumn and adapted for placement on a said column of a fluid test media,said stacked array including in descending sequence:

i. a porous upper glass fiber disc; ii. a porous lower glass fiber disc;iii. a membrane filter disc; and iv. a cellulose acetate disc;

C. said porous glass fiber discs constituting prefilters to removeamorphous matter including white cells from blood to prevent such matterfrom subsequently clogging pores of said membrane;

D. said porous discs constituting dried reagent storage units forelution therefrom by clear filtrate passing through the prefilter disccombination;

E. said membrane filter disc constituting a red blood cell and particleremoval unit from a test specimen;

F. said porous discs constituting fluid volume control units;

and

G. test reagents incorporated in selected ones of said discs andmembrane in said stacked arrays constituting said separate restrictedtest zone areas, operable upon application of whole blood and presetcontrols thereto to visually color indicate the enzyme activity in thewhole blood.

2. in a system as claimed in claim 1, superposed slides mounting saidstacked discs therebetween, said slides being sealed together andconstituting prefabricated test packages.

3. in a system as claimed in claim 2, said discs severally havingselected mutually incompatible in storage reagents required for a giventest impregnated on separate said discs, said reagents contained in saiddiscs being freeze-dried for separation and storage prior to test use.

4. In a system as claimed in claim 3, said upper glass fiber disccontaining Cysteine hydrochloride, said lower glass fiber disccontaining a composite of reagents required for a Creatine phosphokinaseassay excluding N-methylphenazoniurn methosulfate and enzymes, saidmembrane filter disc containing N-methylphenazonium methosulfate, andsaid cellulose acetate disc containing Creatine phosphokinase,Hexokinase and Glucose-fi-phosphate dehydrogenase.

5. In a system as claimed in claim 4, said composite of reagentscomprising in concentration (mg/ml):

Reagents Concentration (mg/ml.) Cystein hydrochloride 1.0 Adenosinediphosphate 1.0

5.0 Ill; and said reagents excluding Cysteine hydrochloride, beingdissolved in glycylglycine buffer, adjusted to pH 6.8.

6. In a system as claimed in claim 5, a blood test specimen placed onsaid upper glass fiber disc in a reagent containing array taking upcysteine hydrochloride, reagents from said lower glass fiber disc, andN-methylphenazonium methosulfate from said membrane filter disc,delivering both filtered plasma and reagents to the enzymes on saidcellulose fiber disc where the reaction takes place.

7 In a system as claimed in claim 5, each said area including a saidstacked array, each said area comprising a spot, three said spotscontaining CPK standards representing normal, elevated, and high enzymelevels, respectively, a fourth said spot being a blank containing allreagents except creatine phosphate and the fifth said spot being adaptedfor assay of CPK and containing reagents for a complete reactionsequence.

8. In a method for quantitating enzyme activity in a system as claimedin claim 7, applying a drop of water to the upper sides of said threestandards containing spots, applying a drop of whole blood to the uppersides of said fourth and fifth spots, inverting the slides mounting saidstacked arrays constituting said spots, observing development of colorand comparing intensity of color developed in said fifth spot with thatin said three standards containing spots for a direct reading of CPKactivity.

9. An enzyme CPK assay test unit for visual determination of enzymecontent of whole blood comprising:

A. a support;

B. means constituting a plurality of test spots mounted on said support:

i. at least first ones of said spots containing reagents for assay ofCPK activity in blood serum including standards representing normal,elevated, and high enzyme levels;

ii. an additional said spot being a blank containing all reagents in thefirst said spots except creatine phosphate; and

iii. a further said spot containing reagents activatable by whole bloodfor a complete reaction sequence;

iv. said reagents being mutually incompatible in storage and separatedby impregnation on a plurality of discrete separate laminae whichcollectively constitute each said spot;

v. said laminae in each said spot being in a rigidly confined stackedarray and some said laminae constituting prefilters to remove amorphousmatter including white cells from blood to prevent such matter fromsubsequently clogging pores of others of said laminae in a said stackedarray.

10. A test unit as claimed in claim 9, said reagents being severallyapplied to discs constituting said laminae fonning said spots toseparate reagents incompatible in storage and being in a freeze-driedstorage condition, the reagents in the first said spots being activatedby water, activation of reagents in all said spots presenting anindicating color therein whereby enzyme content in whole blood isvisually determinable by color comparison with the colors of thestandard indicators.

2. In a system as claimed in claim 1, superposed slides mounting saidstacked discs therebetween, said slides being sealed together andconstituting prefabricated test packages.
 3. In a system as claimed inclaim 2, said discs severally having selected mutually incompatible instorage reagents required for a given test impregnated on separate saiddiscs, said reagents contained in said discs being freeze-dried forseparation and storage prior to test use.
 4. In a system as claimed inclaim 3, said upper glass fiber disc containing Cysteine hydrochloride,said lower glass fiber disc containing a composite of reagents requiredfor a Creatine phosphokinase assay excluding N-methylphenazoniummethosulfate and enzymes, said membrane filter disc containingN-methylphenazonium methosulfate, and said cellulose acetate disccontaining Creatine phosphokinase, Hexokinase and Glucose-6-phosphatedehydrogenase.
 5. In a system as claimed in claim 4, said composite ofreagents comprising in concentration (mg./ml.): Reagents Concentration(mg./ml.) Cystein hydrochloride 1.0 Adenosine diphosphate 1.0Nicotinamide adenine dinucleotide phosphate 1.0 Glucose 3.0Adenosine-5''-monophosphate 4.0 Nitro blue tetrazoleum 1.0 Creatinephosphate 5.0 Magnesium acetate 2.0 N-Methylphenazonium methosulfate 0.1Hexokinase 0.1 IU Glucose-6-phosphate dehydrogenase 5.0 IU Creatinephosphokinase 5.0 IU; and said reagents excluding Cysteinehydrochloride, being dissolved in glycylglycine buffer, adjusted to pH6.8.
 6. In a system as claimed in claim 5, a blood test specimen placedon said upper glass fiber disc in a reagent containing array taking upcysteine hydrochloride, reagents from said lower glass fiber disc, andN-methylphenazonium methosulfate from said membrane filter disc,delivering both filtered plasma and reagents to the enzymes on saidcellulose fiber disc where the reaction takes place.
 7. In a system asclaimed in claim 5, each said area including a said stacked array, eachsaid area comprising a spot, three said spots containing CPK standardsrepresenting normal, elevated, and high enzyme levels, respectively, afourth said spot being a blank containing all reagents except creatinephosphate and the fifth said spot being adapted for assay of CPK andcontaining reagents for a complete reaction sequence.
 8. In a method forquantitating enzyme activity in a system as claimed in claim 7, applyinga drop of water to the upper sides of said three standards containingspots, applying a drop of whole blood to the upper sides of said fourthand fifth spots, inverting the slides mounting said stacked arraysconstituting said spots, observing development of color and comparingintensity of color developed in said fifth spot with that in said threestandards containing spots for a direct reading of CPK activity.
 9. Anenzyme CPK assay test unit for visual determination of enzyme content ofwhole blood comprising: A. a support; B. means constituting a pluralityof test spots mounted on said support: i. at least first ones of saidspots containing reagents for assay of CPK activity in blood serumincluding standards representing normal, elevated, and high enzymelevels; ii. an additional said spot being a blank containing allreagents in the first said spots except creatine phosphate; and iii. afurther said spot containing reagents activatable by whole blood for acomplete reaction sequence; iv. said reagents being mutuallyincompatible in storage and separated by impregnation on a plurality ofdiscrete separate laminae which collectively constitute each said spot;v. said laminae in each said spot being in a rigidly confined stackedarray and some said laminae constituting prefilters to remove amorphousmatter including white cells from blood to prevent such matter fromsubsequently clogging pores of others of said laminae in a said stackedarray.
 10. A test unit as claimed in claim 9, said reagents beingseverally applied to discs constituting said laminae forming said spotsto separate reagents incompatible in storage and being in a freeze-driedstorage condition, the reagents in the first said spots being activatedby water, activation of reagents in all said spots presenting anindicating color therein whereby enzyme content in whole blood isvisually determinable by color comparison with the colors of thestandard indicators.